Asymmetric distribution of pause transfer sequences in apolipoprotein B-100

Abstract

Lipoprotein assembly requires a complex and regulated set of events that includes apolipoprotein B (apoB) translocation across the endoplasmic reticulum (ER) membrane, folding, and association with lipids. Unlike simple secretory proteins which are cotranslationally translocated directly into the ER lumen, nascent apoB contains pause transfer (PT) sequences that direct the transient stopping and subsequent restarting of its translocation, a phenomenon termed translocational pausing. During one particular translocational pause in apoB, the ribosome-membrane junction and ER translocation channel have been shown to be altered in such a way as to expose the nascent polypeptide to the cytosol and direct a change in the proteins neighboring the nascent chain. In this study, we have experimentally identified the location and distribution of the translocational pauses that are present throughout apoB-100. We find that pause transfer sequences are distributed asymmetrically, clustering in three distinct domains: a) nine functional PT sequences appear in the amino terminal 20% of apoB, b) four more PT sequences occur just before the end of apoB-48, and c) an additional ten PT sequences are found between apoB-65-95. These clusters are interrupted by two lipid binding regions of approximately 100 kD each in which no PT sequences occur. The implications of this asymmetric distribution of PT sequences, and their correlation with previously hypothesized structural and functional domains of apoB, are discussed.